PROJECT SUMMARY Although much progress has been made deciphering the effects of anesthetics upon individual ion channels, identification of the neural substrates upon which anesthetics act to produce their behavioral effects remains challenging. Of the key components that characterize the anesthetized state, we focus on volatile anesthetic- induced hypnosis, defined as a lack of awareness to non-noxious stimuli. Understanding how anesthetics produce hypnosis has become more than a central question for neuroscience, as multiple reports over the past decade suggest that existing general anesthetics may annually harm a subset of the 40 million US patients who require anesthesia. One hypothetical alternative to anesthetic-induced unconsciousness is to generate a state of reversible physiological unconsciousness, such as sleep, in which the patient is locked out of access to the state of wakefulness yet recoups restorative benefits of natural sleep. In our original award period, we discovered that volatile anesthetics do directly depolarize a subset of sleep-promoting, preoptic anterior hypothalamic (POAH) neurons. Our lab and others have also shown that every tested general anesthetic (except for ketamine) depolarizes ventrolateral preoptic (VLPO) neurons. However, the exact degree of overlap among neurons triggering endogenous sleep and those activated by anesthetic exposure as well as precise consequences of anesthetic ?hijacking? of endogenous sleep circuitry remain unknown. The central hypothesis of this renewal is that volatile anesthetics will impart a portion of their hypnotic properties by enhancing activity in endogenous POAH neurons, but that key differences in neuronal activation will distinguish endogenous NREM sleep from anesthetic hypnosis. In Aim 1, we will use a novel genetically encoded calcium detector to permanently mark sleep-active POAH neurons in vivo and subsequently determine the exact fraction that is also depolarized by volatile anesthetics. Similarly, we will determine the fraction of anesthetic-depolarized POAH neurons that active during NREM and REM sleep. In Aim 2, we will employ groundbreaking in vivo endoscopic microscopy to visualize and determine real time neuronal activity of POAH neurons as mice cycle naturally across wakefulness, NREM, and REM sleep as well as in these same POAH neurons during a carefully titrated volatile anesthetic exposure. Finally, in Aim 3 we will modulate in vivo activity of sleep-active POAH neurons achieving precise control through neuronal circuit labeling and by recently discovered tissue specific expression markers that distinguish sleep-active neurons from state-indifferent or wake-active neighbors. Conditional expression of virally driven ?designer receptors exclusively activated by designer drugs? (DREADDs) will permit us to depolarize or hyperpolarize POAH neurons to determine the ensuing effects of activating or inhibiting sleep-active POAH neurons upon the stability of the anesthetic state. Cumulatively, these aims will enable us to assess the neuronal convergence/divergence of hypnosis caused by volatile anesthetics and sleep.